Nanofabricated separation matrix for analysis of biopolymers and methods of making and using same

ABSTRACT

Separation matrices useful in the formation of solid-state mm- to cm-scale devices for the rapid, high-resolution separation of single-stranded DNA ladder bands generated by the Sanger dideoxy- or Maxam/Gilbert chemical DNA sequencing procedures are formed from a solid support (1) having a plurality of posts (4) disposed on a first major surface thereof to form an obstacle course of posts (4) and pores (5). The posts are arranged in a regular X, Y array and are separated one from another by a distance of 100 nm or less, preferably 10 to 30 nm, and are optionally separated into lanes 2. The separation matrix can be manufactured by first forming a mold, preferably a reusable mold using lithography techniques. The mold is the reverse of the desired pattern of posts and pores of the obstacle course, and is used for casting the obstacle course. The cast obstacle course is then fused to a solid support and separated from the mold. Alternatively, the separation matrix can be formed from a polymer which undergoes specific and quantifiable swelling in the presence of a selected chemical compound. In this case, the matrix is cast on a mold in a conventional manner with a spacing between posts greater than the desired final spacing of 100 nm or less. For use, a buffer solution saturated with the specific chemical agent that controls swelling is added, causing the posts to swell to a defined amount to achieve the desired separation.

This application claims priority from U.S. Provisional Application Ser.No. 60/000,036 filed Jun. 8, 1995.

BACKGROUND OF THE INVENTION

This application relates to a novel form of separation matrix for theanalysis of biopolymers, particularly nucleic acid polymers.

The use of separation matrices for the analysis of biopolymers is wellestablished. For example, agarose gels, polyacrylamide gel and othertypes of gel matrices are used routinely to separate proteins,polypeptides and polynucieotides into subclasses based upon propertiessuch as size, weight and molecular charge. Analysis of the separatedsubclasses is used to identify and characterize proteins, to detect andcharacterize mutant forms of proteins, and to detect and characterizepolynucleotides. For example, analysis of separated polynucieotidefragments is a basic part of the process for most determinations ofnucleic acid sequence.

Although the gel matrices which have been used for these separations todate are effective and can produce useful analytical results, they arenot without their deficiencies. These deficiencies include (1) arandomness of structure, and (2) a requirement for continuous hydrationafter formation. Both of these deficiencies can lead to unpredictablevariations in the results obtained between one gel and another, whetheras a result of intrinsic variations in the gels, or as a result ofchanges resulting from differing storage conditions. In addition, themicro-inhomogeneity resulting from randomness of the gel structurereduces the actual resolving power of the gels. A further deficiency ofknown gels used in separations of biopolymers arises from the nature ofthe gel materials themselves. The materials used may be subject tobreakdown by high electric fields, thus limiting the field strength thatcould otherwise be employed to obtain more rapid separation. Inaddition, the gel, or materials used in forming the gel such asaccelerators, may interfere with optical detection of the separatedbiopolymers.

Volkmuth et al, Nature 358: 600-602 (1992) have proposed the use of aSiO₂ obstacle course fabricated using optical microlithography for theanalysis of large DNA molecules having a length on the order of 100kilobases. The obstacle course is made up of a regular array of postshaving separations of 1 μm between the posts. DNA loaded onto the arraywas separated by size by the application of an electric field, anddetected using epifluorescence microscopy.

The obstacle course described by Volkmuth et al. is not well-suited foruse in diagnostic applications, however, because the total length of theDNA fragments in most diagnostic DNA sequencing applications, diagnosticRFLP (restriction fragment length polymorphism) procedures, and the likeis between about 20 and 300 to 400 nucleotides. As such, the looping oflong strands of DNA observed by Volkmuth cannot be relied upon as abasis for separation. Furthermore, the fabrication process of Volkmuthet al. uses electron beam lithography to make each individual device,which would be prohibitively expensive for diagnostic applications.

International Patent Publication No. WO94/29707 discloses amicrolithographic array for macromolecule and cell fractionation. Thearray is made using photolithography and then used for separation ofmacromolecules or cells migrating under the influence of an electric,magnetic, hydrodynamic or optical field.

It is an object of the present invention to provide an alternative fromof separation matrix which overcomes these deficiencies of known gelmatrices and which is useful in the analysis of smaller DNA fragmentsuseful in diagnostic applications.

It is a further object of the present invention to provide a separationmatrix for the separation and analysis of biopolymers which has a highlyregular structure.

It is still a further object of the present invention to provide aseparation matrix which can withstand very high electric fieldstrengths.

It is still a further object of the present invention to provide aseparation matrix which permits separated biopolymers to be opticallydetected with very high efficiency.

It is still a further object of the invention to provide a method formanufacturing the novel separation matrices of the invention.

It is still a further object of the present invention to provide amethod for separating biopolymers, and in particular for separatingoligonucleotide fragments for purposes of analyzing DNA sequences, usingthe novel separation matrices of the invention.

SUMMARY OF THE INVENTION

The present invention provides a separation matrix useful in theformation of solid-state mm- to cm-scale devices for the rapid,high-resolution separation of single-stranded DNA ladder bands generatedby the Sanger dideoxy- or Maxam/Gilbert chemical DNA sequencingprocedures. Such device are referred to herein as "DNA SequencingChips."

The separation matrix comprises a solid support having a plurality ofposts disposed on a first major surface thereof to form an obstaclecourse of posts and pores. The posts are arranged in a regular X,Y arrayand are separated one from another by a distance of 100 nm or less,preferably 10 to 30 nm.

The separation matrix of the invention can be manufactured by firstforming a mold, preferably a reusable mold using lithography techniques.The mold is the reverse of the desired pattern of posts and pores of theobstacle course, and is used for casting the obstacle course. The castobstacle course is then fused to a solid support and separated from themold.

Alternatively, the separation matrix can be formed from a polymer whichundergoes specific and quantifiable swelling in the presence of aselected chemical compound. In this case, the matrix is cast on a moldin a conventional manner with a spacing between posts greater than thedesired final spacing of 100 um or less. For use, a buffer solutionsaturated with the specific chemical agent that controls swelling isadded, causing the posts to swell to a defined amount to achieve thedesired separation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C shows a nanofabricated separation matrix accordingto the present invention;

FIG. 2 shows calculated values for DNA chain parameters R and S as afunction of oligonucleotide fragment length, assuming a distance L=0.3nm for adjacent nucleotide residues;

FIG. 3 shows a layered composite useful for forming a mold to make theseparation matrix of the invention;

FIG. 4 shows an apparatus for manufacturing separation matrices;

FIG. 5 shows a layout of electrodes useful in the invention;

FIG. 6 shows a layout of electrodes useful in the invention;

FIG. 7 shows an apparatus in accordance with the invention;

FIG. 8 shows a loading device for loading separation matrices inaccordance with the invention, and

FIG. 9 shows a layout of an integrated DNA diagnostic chip in accordancewith the invention.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1A shows a separation matrix according to the present invention. Asshown, the separation matrix has a solid support 1 on which are disposeda plurality of lanes 2. In the embodiment shown in FIG. 1A, each lane 2is approximately 10 μm wide and 1 μm or less deep, and is separated fromneighboring lanes by a separator 3 of approximately 10 μm in width.(FIG. 1B) The width of each lane, and indeed the use of separated lanesat all is a matter of design choice and is not critical to theinvention, although it should be noted that lane separation allows eachlane to act as a separate capillary. It will be appreciated, however,that the sizes noted above provide for the possibility of 50 lanes in a1 mm wide chip, which allows for very high throughput in a small area.

Each lane 2 contains a plurality of posts 4 which form an obstaclecourse for the separation of the biopolymers as shown in the partial topview of one lane 2 and separator 3 in FIG. 1C. The posts 4 are disposedin a regular pattern leavings pores 5 open for the passage of materialsthrough the separation matrix. The spacing between the posts 4 must belarge enough to permit passage of the biopolymer to be separated, yetsmall enough to provide an obstacle to passage which will result inseparation. The determination of the appropriate size requires aconsideration of many factors, including the overall size of the device,the degree of separation to be achieved by the device, and the size ofthe polymers to be separated. Each line of posts 4 may be in alignmentwith or offset with respect to adjacent lines of posts. It may beadvantageous to form the separation matrix such that the spacing betweenlines of posts is sufficient to allow migrating molecules to reassume arandom distribution of orientations. If spacing is finer, and DNA doesnot achieve random reorientation, reptation behavior result. This wouldbe a different mode of electrophoresis, which may have useful propertiesas well.

As a matter of first principle, the appropriate size for the pores 5between the posts 4 can be determined empirically, i.e., by testing aseries of spacings to determine the effectiveness of each for separationof biopolymeric materials of a given size range, or it can be determinedtheoretically based upon the properties of the polymer in question. Forexample, single-stranded DNA in a denaturing solvent is linear polymerwith a random-coil configuration. For such a polymer, severalstatistical measures of the chain geometry can be defined. Thedisplacement length R is defined as the root-mean-square distancebetween the two terminal groups of the chain. This parameter is directlyrelated to the radius of gyration S, which is defined as theroot-mean-square distance of element i within the polymer from thecenter of gravity of the polymer. According to the derivation given byFlory in 1953, for a linear polymer in a random-coil configuration,

    R=L*(N).sup.1/2

and

    S+2.45*R

where N is the number of elements in the chain, (i.e., the number ofnucleotide residues in a single-stranded oligonucleotide fragment) and Lis the distance between centers of adjacent elements in the chain. FIG.2 shows calculated values for R and S as a function of fragment length,assuming a distance L=0.3 nm for adjacent nucleotide residues.

To use this information to determine the appropriate spacing for aseparation matrix, it is necessary to understand how these parametersare associated with the movement of a polymer through an obstaclecourse. Several recent papers in polymer theory have considered thediffusion of a random coil through a fixed course of obstacles, both inthe presence and absence of a motive force (e.g. an electrophoreticfield). The general conclusion from these studies is that a spacingbetween obstacles that is of the same order of magnitude as thedisplacement length R or the gyration radius S a polymer will imposeentropic barriers to the movement of that polymer though the obstacles.This is the case because the polymer chain will have to transientlyadopt a conformation of low entropy which is thermodynamicallydisfavored to move through the gap.

Applying this conclusion to the separation of oligonucleotide fragmentsused in sequencing, we have assumed that single-stranded fragments ofinterest may have a size in a range from 30 to 1,000 nucleotides. Thiscorresponds to an R value between 1.6 and 10 nm. Therefore, as a firstapproximation, a spacing between obstacles of 1 to 10 nm should beeffective for achieving separation of such fragments. This size range isconsistent with the median pore radius of 0.5 to 4 nm observed forpolyacrylamide gels. Of course, larger spacings will still lead toseparation, although with less efficiency than if a smaller spacing isused, and will be more easily manufactured. Thus, in accordance with oneaspect of the present invention, the spacing between the posts isadvantageously less than 100 nm, preferably 1 to 30 nm. Larger spacingbetween the posts may be particularly useful in a hybrid obstacle courseas discussed more fully below.

Separation matrices according to the invention can be manufactured usingelectron beam lithography to form a mold which is the reverse of thedesired pattern of posts and pores making up the obstacle course,casting the obstacle course in the mask, and then fusing the castobstacle course to a solid support for stripping from the mold.Preferably, the mold is reusable.

To form the mold, a substrate is coated with a pattern receiving layeror layers. For example, as shown schematically in FIG. 3, a tungstensubstrate 31 which will ultimately form the mold, may be coated with athin layer of a dielectric such as silicon dioxide 32, a thin layer of alow cross-section, highly conductive metal, such as aluminum 33, and alayer of an electron sensitive resist 34. Preferred resists are positiveresists such as polymethylmethacrylate derivatives, since these will ingeneral provide the highest resolution. Cowie, J. M. G. Polymers:Chemistry and Physics of Modern Materials, 2nd ed. Blackie, London,(1991). The resist layer is preferably deposited by spray deposition,although formation of a Langmuir/Blodgett film on water, followed bytransfer to a solid surface can also be used for the formation of moldswith resolution on the order of 50 to 80 nm. Miyashita et al., Prog.Polymer Sci. 18: 263-294 (1993).

The coated substrate is positioned on the stage of an electron beamlithography tool having appropriate resolution to draw lines of thedimensions desired. Suitable tools for this purpose include the JEOLJBX-5DII(U) electron beam tool (resolution of 20 nm) and theLeica/Cambridge EBMF 10/5 CS electron beam lithographic instrument(resolution 150 nm). The electron beam is used to write a pattern in theresist which is either a positive image (in the case of a positiveresist which hardens to resist removal when exposed to the electronbeam) or a negative image (in the case of a negative resist whichbecomes removable when exposed to the electron beam) of the posts andpores of the desired obstacle course. The exposed resist is thendeveloped, to leave the patterned mask on the substrate.

Once the mask has been formed, the next step is the transfer of thepattern down through the layers of the substrate using successivetreatments with specific chemical etchants, culminating in the etchingof the substrate itself to produce a reusable mold for use in formingthe separation matrix of the invention.

The separation matrix of the invention can be made of any of a number ofmaterials, and the manner of forming the matrix within the mold willdepend to a large extent on the nature of the material selected. Forexample, separation matrices made from silicon dioxide or similarmaterials can be formed using a chemical vapor deposition process todeposit material within the mold. Thereafter a glass base can be fusedto the deposited materials, for example using the technique of fieldassisted silicon glass fusion (Wallis et al., J. Appl. Phys. 40:3946-3949 (1969)) to increase the dimensional stability of the moldedmatrix and to facilitate its separation from the mold.

The matrix may also be formed from a variety of polymeric materials,including polymethylmethacrylate, ultraviolet-curable polyurethanes,ultraviolet-curable epoxies and other polymers with suitable physicaland chemical properties, i.e., optical transparency and low fluorescenceat relevant wavelengths, thermal conductivity and lack of electriccharge. These materials can be formed into the obstacle course relyingon capillarity as in the techniques of polymer casting (Kim et al,Nature 376: 581-584 (1995), and then cured either chemically orphotochemically.

A mold as described above can also be used to form the separation matrixof the invention using the technique of imprint lithography as describedby Chou et al., Science 272: 85-87 (1996). In this technique, a thinlayer of a polymer resist such as polymethylmethacrylate is applied to asubstrate. The polymer resist is then softened to a gel by heating it toa temperature above its glass transition temperature, and the mold ispressed against the softened resist to form a thickness contrastpattern. The mold is then removed and an anisotropic etching processsuch as reactive ion etching or wet etching is used to remove the resistfrom the compressed areas.

In the case of swellable polymers, larger starting molds can be usedbecause the unswelled spacing between the posts is greater, and thuspresents less of a challenge in terms of molding and separationtechnology. Molds for this purpose can be made by ultravioletlight-based lithographic techniques because less resolution is needed.In this case, the key is to determine the maximum amount which a givenpolymer will swell when in the presence of a saturating amount of theswelling control agent, and then making the posts in a size such thatwhen this amount of swelling occurs, the desired final dimensions areachieved.

Suitable swellable polymers useful in the present invention areinsoluble cross-linked polymers which are modified to have a specificaffinity for a non-DNA material. Such polymers include polyvinylalcohol,polyethyleneimine, polyacrylamide, polystyrene, cross-linked dextran,and polyacrylic acid. Modifiers are selected to provide appropriatespecificity to the polymer such that the amount of swelling can becontrolled by addition of a selected agent. By including a saturatingamount of the selected agent in the electrophoresis buffer, the degreeof swelling of the polymer matrix can be consistently maintained, evenafter cycles of dehydration and rehydration. Synthesis of polymers ofthis type are described in the literature, including in InternationalPatent Publication No. PCT/US91/12626 which is incorporated herein byreference.

After the obstacle course is formed, it is placed on a solid supportwhich will form a part of the final separation matrix, providingdimensional stability to the final product and to the obstacle courseduring separation from the mold. Suitable materials for the solidsupport include fine quartz or pyrex cover slips which can be fused toposts of silicon dioxide and similar materials by field-assisted siliconglass fusion. Wallis et al. J Appl. Phys. 40: 3946-3949 (1969).

After the solid support is fused to the posts of the obstacle course,the separation matrix (support and posts) is separated from the mold. Atthis stage, the separation matrix of the invention can be used forseparation of biopolymers by filling the matrix with a buffer solution,placing opposing ends of the matrix in contact with solution electrodes,loading a sample at one end of the separation matrix, applying anelectric field between the solution electrodes and detecting separatedoligonucleotide fragments at the other end of the separation matrix.There are various nuances which can be employed, however, to improve theversatility and utility of the separation matrix of the invention.

FIG. 4 shows an apparatus suitable for forming multiple replicate copiesof a DNA sequencing chip, using a mold and a photopolymerizable materialusing the procedures discussed above. As shown, a moveable base 41having a lamp 42 for delivering light suitable for inducingphotopolymerization positioned therein is disposed within a housing 43.The base 41 supports a chemically treated glass cover slip or othersubstrate 44 in alignment with an injection port 45 in the housing.Monomer or prepolymer solution is injected through the injection port 45onto the top of the substrate 44. A piston 46 having the mold 47 for thepattern of plugs and pores is then pressed down into the solution on topof the substrate 44. The lamp 42 is then turned on to polymerize thesolution in the mold, after which the molded polymer is separated fromthe mold 47 and the housing 43 by raising the piston 46 and lowering thebase 41.

A modified version of the device of FIG. 4 could instead be used torepetitively make multiple copies of a DNA sequencing chip by imprintlithography. In this case, instead of a monomer or prepolymer solution,a polymeric sheet would be used as the starting materials. This would beheated above the glass transition temperature and then piston 46 havingmold 47 would be pressed down into the softened polymeric sheet to makethe replica. The temperature would be lowered again below the glasstransition point and then piston 46 and mold 47 would be removed.Instead of lamp 42, this apparatus would have a heating element.

In addition to the posts making up the separation matrix, DNA SequencingChips in accordance with the invention include electrodes deposited on abottom or top substrate to generate an electric field to inducemigration of materials in the matrix. This can be a simple pair ofelectrode disposed at opposing ends of the separation matrix to imposean electric field in one direction on materials within the separationmatrix. Preferably, however, an extended series of micro-electrodes isdisposed on the substrate, multiple electrodes within each lane of thedevice.

FIG. 5 illustrates one arrangement of micro-electrodes and conductingwires on the substrate with posts and pores omitted for clarity. Themicro-cathodes, 102, and micro-anodes, 103, are deposited in the lanes2. Conducting leads 105 can be formed directly across the substrate 101or if micro-holes are cut through the substrate, it is possible toconstruct the wire connections, 116, on the underside of the substrate1, as illustrated in FIG. 6. The micro-electrodes and leads may becompositions of any relatively high conductance materials. Preferablythe electrodes will be made of a coated or corrosion resistant materialsuch as the noble metals platinum or gold. Corrosion resistancesufficient to withstand exposure to the buffer system employed in theseparation is required, although noble metals are not necessary if thedevice is disposable.

The pattern of the micro-electrodes on the substrate may be chosenaccording to the demands of the application for which the invention isemployed. Two type of layouts are generally available. The first,illustrated in FIG. 5 is a spatially dispersed array in which eachmicro-electrode can be activated separately. Thus, electrodes which arerequired for a certain application may be turned on, while unnecessaryones may simply be turned off. The second option is a dedicated patternwhich is employed for applications where known separation distances andvoltages are available. In this case, the electrodes can be laid out ina fixed pattern which is known to be satisfactory for the desiredapplication.

To use the separation matrix of the present invention as a DNASequencing chip, the pores of the separation matrix are first filledwith a separation fluid. This may be a buffer, a buffer containing aswelling agent, or it may be a suspension of a secondary obstacle whichacts in concert with the posts of the separation matrix to form a hybridseparation matrix. In general, the secondary obstacles will be ahomogeously sized and distributed suspension of structures having a sizeon the same order as the radius of gyration of the polymers to beseparated, i.e., about 0.5 to 5 times the radius of gyration. Thesecondary obstacles should also be compatible with the detection systemused. Thus, for fluorescence detection the secondary obstacles should betransparent or translucent, with little or no fluorescence at thewavelengths of interest. Materials which can be used as secondaryobstacles include monodisperse microspheres as described by Hosaka etal, Polym. Int. 30: 505-511 (1993) and Sanghvi et al, J. Microencaps.10: 181-194 (1993), water-soluble fullerenes (C60) as described inDiederich et al., Science 271: 317-323 (1996), and self-assemblingdendrimer as described in Newkome et al, J. Org. Chem. 58: 3123 (1993)and Zimmerman et al., Science 271: 1095-1098 (1996).

The sample to be evaluated, particularly a sample of a nucleic acidsequencing mixture prepared by the Sanger or Maxam/Gilbert methods isthen loaded at one end of the separation matrix, for example in anintegrated apparatus as shown schematically in FIG. 7. Preferably, aplurality of samples are loaded, one to each lane of the separationmatrix.

The minimum practical loading volume, using a commercially-availablemicropipette, is around 100 nL (Hamilton, 1993). In contrast, a stackedelectrophoresis zone 10 μm wide×1 μm deep×10 μm high (as in theentrance-way to a capillary channel of FIG. 1) will occupy a volume of0.1 pL. Therefore a very substantial reduction in volume (up to 16⁶-fold) may be required during the initial stacking process. Suchhigh-efficiency stacking during the initial phase of electrophoresis canbe achieved with the loading device 301 shown in more detail in FIG. 8.

The device of FIG. 8 is shown in an orientation appropriate for loadingsample into a channel of a DNA sequencing chip 203 that is in thevertical orientation. The loader consists of a large rectangular channel201 attached at right angles to a second smaller rectangular channel202. The upper face of the large rectangular channel 201 is open, andreceives a volume (for example 100 nL of unconcentrated samplecontaining a DNA mixture to be separated. The lower face The lower faceof the smaller rectangular channel 202 is also open and releases theconcentrated sample (approx 1 pL) into one of the functional channels ofthe DNA sequencing chip 203. There is an unrestricted passageway betweenthe large and small channels, to allow sample to flow between them, at atime after a first concentration step and before a second concentrationstep.

In the first concentration step, sample loaded into the top of the largechannel 202 is electrophoresed using a filed generated betweenelectrodes 205a and 205b. The DNA is collected on a semipermeablemembrane 204a which has a molecular weight cutoff low enough to preventpassage of the DNA but which permits passage of the solvent from thesample, thereby effecting a first concentration of the sample on thesemi-permeable membrane 204a.

Next, a second set of electrodes 206a and 206b are turned on to generatecause the concentrated sample to migrate in a direction perpendicular tothe original migration from the semi permeable membrane 204a into thesmall channel 202. A second semipermeable membrane 204b retains samplewithin the small channel 202 while permitting passage of solvent.Finally, a third electrode set 207a and 207b is used to electrophoresethe doubly-concentrated sample from the small channel 202 into one ofthe functional channels of the DNA sequencing chip 203.

If is also possible by means of thermocouple strips 208 disposed aboutthe periphery of the large channel 201 to achieve localized cooling ofbuffer in the large channel, via the Peltier effect. This strategy canbe used to lower the temperature of a glycerol-containing buffer belowthe glass-transition temperature, thus creating a viscosity trap closeto the semipermeable membrane 204a which prevents back-diffusion ofconcentrated DNA in the vertical direction within the large channelafter the first concentration step.

It is also possible to use a simplified version of the device of FIG. 8,in which only a single stacking operation is employed. This simplifieddevice consists of channel 201, semipermeable membrane 204a, electrodesets 205a and 205b, and 206a and 206b, and optionally the Peltier strips208. Electrode set 206a and 206b causes the concentrated sample to beelectrophoresed directly into a DNA sequencing chip rather than into asecond concentration channel.

The processes that occur during the loading operation can be summarizedas follows: (1) electrophoresis along the z-dimension onto asemipermeable membrane; (2) optional trapping of sample close to themembrane with a "viscosity trap"; (3) electrophoresis in the x-directioninto a second semipermeable membrane; and electrophoresis in thez-direction into a functional channel in the DNA sequencing chip.

The degree of sample concentration that can be achieved in either of thestacking steps can be calculated from the theory of equilibriumelectrophoresis using the equation:

    C(z)=C.sub.0 e.sup.σ(z-z.sbsp.0.sup.)-2B[C(z)-C.sbsp.0.sup.]

where C₀ =concentration at arbitrary reference distance (z₀) along theelectrophoretic direction, B=second virial coefficient, and σ=E·[ψ/k_(B)T] where E=electric field strength (V/cm), ψ=apparent or effective netcharge (Coulombs), k_(B) =Boltzmann's constant, and T=absolutetemperature. It is apparent from this equation that an arbitrarily largedegree of stacking or concentration can be achieved simply by raisingthe electric filed strength to a sufficiently high value.

Once the samples are loaded, an electric field is applied to thesequencing chip 302 placed within holder 303 using power supply 305 toinduce migration of the sample within the separation matrix. The fieldmay be constant, or periodic field inversions can be used in increasethe resolution of the single-stranded DNA ladder bands. Current can alsobe applied to successive electrodes along the length of the lane, or canbe applied to parallel lines of electrodes within a lane to inducetwo-dimensional separation within the lane.

After separation of the sample into ladder bands, the DNA is detected ata detection site. The detector 304 may be of any type, and will varydepending on the nature of the material being detected. When thesequencing reactions utilize a 5'-fluorescently labeled sequencingprimer, the separated bands can be detected by fluorescence. Threealternative illumination and detection schemes are exemplary of systemswhich can be used.

(i) Diode laser illuminator and fibre optic/phototransistor detector(Sepaniak et al., J. Microcolumn Separations 1: 155-157 (1981); Foret etal., Electrophoresis 7: 430-432 (1986); Hirokawa et al., J.Chromatography 463: 39-49 (1989); U.S. Pat. No. 5,302,272).

(ii) Array of diode-laser illuminators and two-dimensional CCD detector.The design for a two-dimensional CCD array detector has been describedelsewhere (U.S. Pat. Nos. 4,874,492 and 5,061,067 which are incorporatedherein by reference, Eggers et al., BioTechniques 17: 516-524 (1994);Lamture et al., Nucleic Acids Res. 22: 2121-2125 (1994)). Such a systemis advantageously constituted as a fixed array, into which aboard-mounted DNA sequencing chip is inserted. This separates themass-produced and relatively inexpensive sequencing chip from theexpensive detection system.

(iii) Epifluorescence Microscope and Image Intensifying Camera.

While fluorescence detection is the most common technique currentlyemployed in analysis of DNA sequencing fragments, and thus is apreferred approach in the present invention, other detector types can beused. For example, a subject molecule labeled with a radioactive moietymay be detected with a radiation detector, such as X-Ray film or ascintillation counter. Unmodified nucleic acids may also be detected byshifting polarization of input radiation as disclosed in U.S. patentapplication Ser. No. 08/387,272, which application is incorporatedherein by reference.

In the most general application, the invention is used to move and/orseparate species of charged molecules, and in particular chargedbio-molecules such as proteins and nucleic acids. Further, the instantinvention may be used to purify one molecular species from a sample ofmixed molecular species. The object of combining the post and poreseparation matrix with closely positioned electrodes is the creation oflocalized very-high density electric fields in the separation matrix.These localized fields can be supported with very low energy powersupplies, and are therefore energy efficient. U.S. patent applicationSer. No. 08/332,577 which is incorporated herein by referencedemonstrates the usefulness of high density electric fields forseparating charged molecules such as nucleic acids and proteins. Nucleicacids separated under extremely high electric fields (100-400 V/cm) overlocalized areas (1-5 mm²) can be detected and/or used by conventionaldetectors, biosensors and micro-reactor components. These miniaturecomponents may be located at one site, or more than one site on theapparatus.

While simple separation and detection of DNA sequencing fragments is apreferred application of the chips of the invention, the chips can alsobe used to perform more complicated and complete analysis. Thus, thechips of the invention can be fabricated so as to move samples from onewell to another for different treatments, such as in an integrated DNAdiagnostic chip.

As described in Published PCT patent applications Nos. WO 96/07761 andWO 96/01908, which are incorporated herein by reference, the most costeffective means of DNA diagnosis involves a hierarchical method oftesting, wherein a series of analyses of increasing accuracy areemployed to diagnose the presence or absence of genetic mutation. It ispossible to integrate all the hierarchical steps on one chip, asillustrated in FIG. 9.

FIG. 9 shows an integrated DNA diagnostic chip which uses the diagnosticmethod of the above noted patent application. A highly simplifieddiscussion of the steps involved in the hierarchical method is providedbelow. In the first step, a group of exons of a gene to be diagnosed isexamined for insertion and deletion mutations. In a reaction tube (notshown) the suspect exons of the patient are amplified by multiplexed PCRusing oligonucleotide primers labeled with a fluorophore (not shown).The resulting fragments of DNA are loaded into the separation matrix,801 with a loader, 802, at loading site, 803. Upon activating a group ofmicroclectrodes, 804, the sample will migrate through the separationmatrix, 801, and resolve into discrete bands of distinct species, 805,806 and 807. A laser source, 808, of a wavelength suitable to excite thefluorophore is directed to an excitation site, 809. As a band, 806,passes through the excitation site, 809, its fluorescence emissions aredetected by detector 810. The fluorescence emissions may be recorded ordisplayed, 811. If the band has an insertion or deletion relative to thenormal gene, it will pass through the excitation zone at a timedifferent from the expected time. Such a difference indicates thepresence of a mutation in that fragment. The difference can be directlyreported to the patient file. If, however, no insertion or deletionmutation is found, it is then necessary to turn to the second step ofthe diagnostic method, and determine the actual sequence of the DNA todetermine the presence or absence of point mutations. To achieve thisobjective, a separated DNA fragment is moved, according to the method ofthe instant invention, by a series of micro-electrodes, 812 to areaction center, 813.

Immobilized enzymes such as DNA sequencing enzymes may be located atreaction pools, as described in U.S. Pat. Nos. 4,975,175 and 5,286,364,which are incorporated herein by reference. When provided with properreagents, which may be added by a second capillary pipettor, 814, thesequencing reaction may be carried out in situ on the diagnostic chip.After a suitable length of time, the reaction is completed. Thesequenced DNA sample is then separated in a third direction by theactivation of a third group of micro-electrodes, 815. The DNA sequenceis obtained according to conventional fluorescence DNA sequencing: Thelaser source, 808, is directed to the excitation site, 816. Fluorescenceemissions of the sample are recorded by a detector, 817 and recorded ordisplayed, 818. The presence or absence of point mutations is thenrecorded and reported to the patient file.

What is claimed is:
 1. A separation matrix comprising a substrate havingdisposed on a first major surface thereof a plurality of posts, saidposts being arranged at regular intervals in a plurality of parallellines, wherein the interval between adjacent posts in a line is 100 nmor less, characterized in that the area between the posts is filled witha secondary obstacle which increases the challenge posed to a chargedmolecule migrating through the matrix.
 2. The separation matrixaccording to claim 1, wherein the interval between adjacent posts in aline is 30 nm or less.
 3. The separation matrix according to claim 1,wherein the posts are divided into a plurality of lanes orthogonal tothe parallel lines, each lane being separated from adjacent lanes by aregion of substrate having no posts thereon.
 4. A chip forelectrophoretic separation of charged polymers comprising(a) asubstrate; (b) a plurality of posts, said posts being arranged atregular intervals in a plurality of parallel lines on a first majorsurface of the substrate, wherein the interval between adjacent posts ina line is 100 nm or less; and (c) at least two electrodes disposed onthe substrate to make electrical contact with a liquid medium disposedin an area surrounding the posts for inducing an electric field for theelectrophoretic separation of charged polymers, characterized in thatthe area between the posts is filled with a secondary obstacle whichincreases the challenge posed to a charged molecule migrating throughthe matrix.
 5. The chip according to claim 4, wherein the intervalbetween adjacent posts in a line is 30 nm or less.
 6. The chip accordingto claim 4, wherein the posts are divided into a plurality of lanesorthogonal to the parallel lines, each lane being separated fromadjacent lanes by a region of substrate having no posts thereon.
 7. Thechip according to claim 4, wherein an X-Y array of electrodes isdisposed on the substrate.
 8. A method for separating a mixturecontaining a plurality of species of biopolymers into subclasses ofbiopolymers comprising the steps of loading the mixture onto aseparation matrix and applying an electric field to cause chargedbiopolymers in the mixture to migrate and be separated, wherein theseparation matrix is a chip comprising a substrate having disposed on afirst major surface thereof a plurality of posts, said posts beingarranged at regular intervals in a plurality of parallel lines, whereinthe interval between adjacent posts in a line is 100 nm or less,characterized in that the area between the posts is filled with asecondary obstacle which increases the challenge posed to a chargedmolecule migrating through the matrix.
 9. The method according to claim8, further comprising the step of detecting the separated biopolymerswithin the chip.
 10. The method according to claim 9, wherein theseparated biopolymers are detected using a fluorescence detector.
 11. Amethod for sequencing nucleic acids comprising the steps of loading amixture containing products from a sequencing reaction onto a separationmatrix, applying an electric field to cause the products from thesequencing reaction in the mixture to migrate and be separated, anddetecting separated bands of sequencing reaction products, wherein theseparation matrix is a chip comprising a substrate having disposed on afirst major surface thereof a plurality of posts, said posts beingarranged at regular intervals in a plurality of parallel lines, whereinthe interval between adjacent posts in a line is 100 nm or less,characterized in that the area between the posts is filled with asecondary obstacle which increases the challenge posed to a chargedmolecule migrating through the matrix.
 12. The method according to claim11, wherein the separated bands are detected using a fluorescencedetector.
 13. An apparatus for separation of plurality of species ofbiopolymers into subclasses of biopolymers comprising(a) a holder forreceiving a chip for electrophoretic separation of charged polymers,said chip comprising(1) a substrate; (2) a plurality of posts, saidposts being arranged at regular intervals in a plurality of parallellines on a first major surface of the substrate, wherein the intervalbetween adjacent posts in a line is 100 nm or less; and (3) at least twoelectrodes disposed on the substrate to make electrical contact with aliquid medium disposed in an area surrounding the posts for inducing anelectric field for the electrophoretic separation of charged polymers,said chip having a fluid medium contained in the area between the posts;and (b) a power supply for applying an electric field to the chip tocause charged biopolymers in the mixture to migrate within the chip andbe separated, characterized in that the apparatus further comprises aloader for decreasing the volume of a sample to be loaded onto a chipdisposed within the holder.
 14. The apparatus according to claim 13,further comprising a detection system for detecting charged separatedbiopolymers within a chip disposed in the holder.
 15. The apparatusaccording to claim 14, wherein the detection system is a fluorescencedetection system.
 16. An apparatus for use in a method for sequencingnucleic acids, comprisinga separation matrix in the form of a chipcomprising a substrate having disposed on a first major surface thereofa plurality of posts, said posts being arranged at regular intervals ina plurality of lines, wherein the interval between adjacent posts in aline is 100 nm or less, characterized in that the area between the postsis filled with a secondary obstacle which increases the challenge posedto a charged molecule migrating through the matrix; at least twoelectrodes disposed on the surface to make electrical contact with aliquid medium disposed in an area surrounding the posts for inducing anelectric field for the separation of charged polymers; a power supplyfor applying an electric field to the chip; and a loader for loading asample onto the separation matrix, wherein the loader comprises a firstconcentration channel, a first pair of electrodes, a first semipermeablemembrane effective to retain polynucleotide fragments while allowingpassage of smaller molecules, and a second pair of electrodes, whereinthe first semipermeable membrane is located between the first pair ofelectrodes whereby polynucleotide fragments migrating in a firstelectric field generated within the first concentration channel betweenthe first pair of electrodes are retained on the first semipermeablemembrane, and wherein the second pair of electrodes generate a secondelectric field perpendicular to the first electric field for migratingthe retained polynucleotide fragments from the first semipermeablemembrane.
 17. The apparatus according to claim 16, further comprising asecond concentration channel, a second semipermeable membrane effectiveto retain the biopolymers while allowing passage of smaller molecules,and a third pair of electrodes, said second pair of electrodes causingthe retained biopolymers to migrate from the first semipermeablemembrane through the second concentration channel to the secondsemipermeable membrane, and said third set of electrodes generating athird electric field perpendicular to the second electric field formigrating retained biopolymer from the second semipermeable membrane.18. The apparatus according to claim 16, further comprising means forlowering the temperature in a portion of the first concentration channelto form a viscosity trap.
 19. The apparatus according to claim 18,wherein the means for lowering the temperature in a portion of the firstconcentration channel is a thermocouple strip.
 20. An apparatus forseparation of plurality of species of biopolymers into subclasses ofbiopolymers comprising:(a) a holder for receiving a chip forelectrophoretic separation of charged polymers, said chip comprising(1)a substrate; (2) a plurality of posts, said posts being arranged atregular intervals in a plurality of parallel lines on a first majorsurface of the substrate, wherein the interval between adjacent posts ina line is 100 nm or less; and (3) at least two electrodes disposed onthe substrate to make electrical contact with a liquid medium disposedin an area surrounding the posts for inducing an electric field for theelectrophoretic separation of charged polymers, said chip having a fluidmedium contained in the area between the posts; and (b) a power supplyfor applying an electric field to the chip to cause charged biopolymersin the mixture to migrate within the chip and be separated, and (c)means for receiving a sample, decreasing the volume of the sample toincrease the concentration of the biopolymers in the sample and loadingthe concentrated sample onto the chip.
 21. The apparatus according toclaim 20, further comprising a detection system for detecting separatedbiopolymers within a chip disposed in the holder.
 22. The apparatusaccording to claim 21, wherein the detection system is a fluorescencedetection system.